A high throughput screen for X-reactivation and reprogramming by small molecules X chromosome inactivation (XCI), the process by which female mammals (XX) balance X-linked gene expression compared to males (XY), is initiated by the expression of the non-coding RNA Xist and encompasses a series of chromatin changes that establishes and maintains the silent state of the inactive X. This repressive environment is reversed by a form of epigenetic reprogramming known as X-reactivation, which occurs in vivo in the early embryo and the germ line, or in vitro during the formation of induced pluripotent stem (iPS) cells. While XCI has been intensively studied for many years and has yielded insight into the general mechanisms of gene regulation, much less is known about X-reactivation. We propose that identifying new molecules that target X-reactivation in mouse cells will also prove useful for reprogramming iPS cells. In contrast to mouse iPS cells, most human iPS cell lines fail in X-reactivation, and for future plans after this proposed project, we look forward to testin new small molecule probes on human iPS cells. The generation of iPS cells has transformed our understanding of the interplay between genetics and epigenetics and has tremendous therapeutic potential. As currently practiced, this process is inefficient and not always capable o generating cells that are truly equivalent to pluripotent embryonic stem (ES) cells. A few small molecules, including the DNA-methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-dC), have been identified that partially de-repress genes on the inactive X in somatic cells and that also aid the formation of iPS cells, further underscoring the tight relationship between XCI and iPS cell reprogramming. A female mouse fibroblast cell line carrying an X-linked GFP reporter gene will be used to study X- reactivation. Application of the positive control 5-aza-dC causes dose-dependent reactivation of X-GFP. For the high-throughput screen (HTS), GFP fluorescence resulting from the application of test compounds will be assayed by automated microscopy. A secondary screen will test the reactivation of specifically the inactive alleles of four endogenous X-linked genes. Probes emerging from these screens will be useful tools to dissect X-reactivation and perhaps shed further light on XCI as well. In a final assay, X-reactivation probes will be tested for their ability to enhance the reprogramming of fibroblasts into iPS cells. While most high throughput screens target a specific enzyme or pathway, the primary screen proposed here is based on a biological outcome that is the result of many epigenetic mechanisms that are as yet incompletely understood. The advantage of this approach is that we expect to identify probes of novel proteins that play crucial roles in maintaining the inactive state of the X-chromosome, that are required for X- reactivation, and that are also relevant to reprogramming.